Tricyclic nucleosides and oligomeric compounds prepared therefrom

ABSTRACT

The present invention provides novel tricyclic nucleosides and oligomeric compounds prepared therefrom. Incorporation of one or more of the tricyclic nucleosides into an oligomeric compound is expected to enhance one or more properties of the oligomeric compound. Such oligomeric compounds can also be included in double stranded compositions. In certain embodiments, the oligomeric compounds provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

FIELD OF THE INVENTION

The present invention relates to tricyclic alkyl-substituted nucleosidesdescribed by the general Formula I and oligomeric compounds preparedtherefrom.

BACKGROUND OF THE INVENTION

Antisense technology is an effective means for reducing the expressionof specific gene products and can therefore be useful in therapeutic,diagnostic, and research applications. Generally, the principle behindantisense technology is that an antisense compound (a sequence ofoligonucleotides or analogues thereof) hybridizes to a target nucleicacid and modulates gene expression activities or function, such astranscription and/or translation. Regardless of the specific mechanism,its sequence-specificity makes antisense compounds attractive as toolsfor target validation and gene functionalization, as well astherapeutics to selectively modulate the expression of genes involved inthe pathogenesis of diseases.

Chemically modified nucleosides are routinely incorporated intoantisense compounds to enhance its properties, such as nucleaseresistance, pharmacokinetics or affinity for a target RNA.

Chemical modifications have improved the potency and efficacy ofantisense compounds, improving their potential for oral delivery orsubcutaneous administration, or decreasing their potential for sideeffects. Chemical modifications increasing potency of antisensecompounds allow administration of lower doses, which reduces thepotential for toxicity. Modifications increasing the resistance todegradation result in slower clearance from the body, allowing for lessfrequent dosing. The synthesis of tricyclic nucleosides (Steffens etal., Helvetica Chimica Acta, 1997, 80, 2426-2439) and theirincorporation into oligomeric compounds has been reported in theliterature (Steffens et al., J. Am. Chem. Soc., 1997, 119, 11548-11549;Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; Renneberg etal., J. Am. Chem. Soc., 2002, 124, 5993-6002; Scheidegger et al., Chem.Eur. J., 2006, 12, 8014-8023). Fully modified tricyclic oligonucleotideswere shown to be more stable against nucleolytic degradation in fetalcalf serum compared to unmodified oligodeoxynucleotides and to producebiological antisense effects in cellular assays, such as splicerestoration of mutant β-globin (Renneberg et al., Nucleic Acids Res.2002, 30, 2751-2757); or exon skipping in cyclophilin A (Ittig et al.,Nucleic Acids Research, 2004, 32, 346-353).

BRIEF SUMMARY OF THE INVENTION

Provided herein are tricyclic nucleosides having Formula I andoligomeric compounds prepared therefrom. More particularly, tricyclicnucleosides having Formula I are useful for incorporation at one or morepositions of an oligomeric compound. In certain embodiments, theoligomeric compounds provided herein are characterized by one or moreenhanced properties such as nuclease stability, cell permeability,bioavailability or toxicity. In certain embodiments, the oligomericcompounds as provided herein hybridize to a portion of a target RNAresulting in loss of normal function of the target RNA. The oligomericcompounds provided herein are also useful as primers and probes indiagnostic applications. In certain embodiments, oligomers comprisingtricyclic nucleosides provided herein show significantlyimproved—compared to unmodified DNA or RNA oligomers—cellar uptakeindependent of transfection reagents such as liposomes. In certainembodiments, oligomers comprising tricyclic nucleosides provided hereinshow significantly increased—compared to unmodified DNA or RNAoligomers—thermal stability (duplex melting point).

The variables are defined individually in further detail herein. It isto be understood that the tricyclic nucleosides having Formula I and theoligomeric compounds provided herein include all combinations of theembodiments disclosed and variables defined herein.

According to a first aspect of the invention, a tricyclic nucleoside isprovided that is described by the general Formula I:

wherein:

-   -   Bx is a heterocyclic base moiety;    -   one of T¹ and T² is hydroxyl (—OH) or a protected hydroxyl and        the other of T¹ and T² is a phosphate or a reactive phosphorus        group, with T¹ optionally being a solid support for        oligonucleotide synthesis;    -   q¹ and q² are each, independently, H, F or Cl,    -   at least one of q³, q⁴ and q⁵ is, independently, a group        described by a general formula -A¹-X_(h)-A²-Y_(n), wherein        -   A¹ is a C_(k)-alkyl, C_(k)-alkenyl or C_(k)-alkynyl, with k            being an integer selected from the range of 0 to 20,        -   X_(h) is —C(═O)—, —C(═O)O—, —C(═O)NR—, —O—, —S—, —NR—,            —C(═O)R, —C(═O)OR, —C(═O)NR₂—, —OR, —SR or —NR₂, with each R            being selected independently from H, methyl, ethyl, propyl,            butyl, acetyl and 2-hydroxyethyl, and h is 0 or 1,        -   A² is a C_(i)-alkyl, C_(i)-alkenyl or C_(i)-alkynyl, with i            being an integer selected from the range of 0 to 20,        -   Y is a substituent group attached to any carbon atom on A¹            and/or A², selected from —F, —Cl, —Br, ═O, —OR, —SR, —NR₂,            —NR₃+, NHC(═NH)NH₂, —CN, —NC, —NCO, —NCS, —SCN, —COR, —CO₂R,            CONR₂, —R, with each R being selected independently from H,            methyl, ethyl, propyl, butyl, acetyl and 2-hydroxyethyl, and            n is 0, 1, 2, 3, 4, 5 or 6,        -   wherein k+i equals at least 1,        -   and wherein any —OR, —SR, —NR₂, —CO₂R, CONR₂ for which R is            H may optionally be protected by a protecting group used in            solid phase oligonucleotide chemistry,    -   the other ones of q³, q⁴ and q⁵ are, independently, H, F or Cl,    -   one of z¹ and z² is H and the other of z¹ and z² is H, —OH, F,        Cl, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂.

One of ordinary skill in the art of organic chemistry understandsthat—concerning the selection of oxygen (═O) as a substituent group—theoxygen atom is attached via a double bond to any carbon atom on A¹ orA². A C_(k)-alkyl, C_(k)-alkenyl or C_(k)-alkynyl in the context of thepresent specification refers to an alkyl, alkenyl or alkynyl moiety,respectively, having k carbon atoms in a linear chain. The index i of A²is applied similarly. It is understood that embodiments for which k is0, A¹ is not present, and for embodiments for which i is 0, A² is notpresent. If n substituent groups are present, these can be present bothon A¹ and A².

In some embodiments, Bx is a pyrimidine, substituted pyrimidine, purineor substituted purine. In some embodiments, Bx is selected from uracil,thymine, cytosine, 5-methylcytosine, adenine and guanine. In someembodiments, Bx is an aromatic heterocyclic moiety capable of formingbase pairs when incorporated into DNA or RNA oligomers in lieu of thebases uracil, thymine, cytosine, 5-methylcytosine, adenine and guanine.

In some embodiments, T¹ is hydroxyl or protected hydroxyl, and T² is areactive phosphorus group selected from an H-phosphonate or aphosphoramidite. In some embodiments, T¹ is a triphosphate group and T²is OH. In some embodiments, T¹ is 4,4′-dimethoxytrityl and T² isdiisopropylcyanoethoxy phosphoramidite. In some embodiments, T¹ is acontrolled pore glass surface. According to certain embodiments of thisembodiment, T¹ is a long chain alkylamine controlled pore glass surfaceor similar solid phase support used in oligonucleotide solid phasesynthesis, to which a 3′-O-succinylated nucleoside is linked via anamide function.

In some embodiments, one of z¹ and z² is F, OCH₃ or O(CH₂)₂—OCH₃. Insome embodiments, one of z¹ and z² is F. In some embodiments, one of z¹and z² is F and the other one is H. In some embodiments, z¹ and z² areeach H. In some embodiments, z¹ and z² are each F. In some embodiments,z¹ and z² are each H.

In some embodiments, q¹ and q² are H. In some embodiments, one of q¹ andq² is F and the other one is H.

In certain embodiments, the tricyclic nucleoside carries in position q³,q⁴ or q⁵ one substituent having 3 to about 18 carbon atoms, optionallywith a cationic group on the chain. Such substituents are useful forincorporation into oligonucleotides that, as a function of the alkylsubstituent chain length and substitution and the number of suchmodified nucleotides, provide improved transport of the oligonucleotidein the body, and improved cellular uptake. Without wishing to be boundby theory, the inventors hypothesize that the observed behavior of suchmodified oligonucleotides may at least partially be explained byself-aggregation of the oligonucleotides by hydrophobic interaction.

EMBODIMENTS A

In some embodiments, the substituent is a C₃ to C₁₆ alkyl moiety, i.e. kis 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, h is 0, i is 0 andn is 1, 2, or 3. In a subgroup thereof, a C₃ to C₁₆ alkyl moiety is inposition q³; q⁴ and q⁵ are H, and optionally, one of q¹ and q² is H andthe other one is F or Cl, or both of q¹ and q² are H. In anothersubgroup thereof, a C₃ to C₁₆ alkyl moiety having 1-6 substituents is inposition q⁴ or q⁵, the other one of q⁴ and q⁵, and q³, are H, andoptionally, one of q¹ and q² is H and the other one is F or Cl, or bothof q¹ and q² are H.

EMBODIMENTS B

In some embodiments, the substituent is an acetic acid C₃ to C₁₆ amideor ester, i.e. k is 1, h is 1 and X is COO—, CONH— or CONR—, i is 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 and n is 1, 2, or 3. In asubgroup thereof, an acetic acid C₃ to C₁₆ amide or ester is in positionq³; q⁴ and q⁵ are H, and optionally, one of q¹ and q² is H and the otherone is F or Cl, or both of q¹ and q² are H. In another subgroup thereof,an acetic acid C₃ to C₁₆ amide or ester is in position q⁴ or q⁵, theother one of q⁴ and q⁵, and q³, are H, and optionally, one of q¹ and q²is H and the other one is F or Cl, or both of q¹ and q² are H.

EMBODIMENTS C

In some embodiments, the substituent is an C₃ to C₁₆ alkoxy moiety, i.e.k is 0, h is 1 and X is —O—, i is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16 and n is 1, 2, or 3. In a subgroup thereof, a C₃ to C₁₆alkoxy moiety is in position q³; q⁴ and q⁵ are H, and optionally, one ofq¹ and q² is H and the other one is F or Cl, or both of q¹ and q² are H.In another subgroup thereof, a C₃ to C₁₆ alkoxy moiety is in position q⁴or q⁵, the other one of q⁴ and q⁵, and q³, are H, and optionally, one ofq¹ and q² is H and the other one is F or Cl, or both of q¹ and q² are H.

EMBODIMENTS D

In some embodiments, q³ is the group described by the general formula-A¹-X_(h)-A²-Y_(n), k and i independently are 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15 and the sum of k and i is at least 3 and nomore than 17, h is 1 and X is selected from COO—, CONH—, CONR—, —O—, andCO, and n is 0, 1, 2, or 3. In a subgroup thereof, q⁴ and q⁵ are H, andoptionally, one of q¹ and q² is H and the other one is F or Cl, or bothof q¹ and q² are H.

EMBODIMENTS E

In some embodiments, either of q⁴ or q⁵ is the group described by thegeneral formula -A¹-X_(h)-A²-Y_(n), k and i independently are 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 and the sum of k and i isat least 3 and no more than 17, h is 1 and X is selected from COO—,CONH, CONR—, —O—, and CO, and n is 0, 1, 2, or 3. In a subgroup thereof,the other one of q⁴ and q⁵, and q³, are H, and optionally, one of q¹ andq² is H and the other one is F or Cl, or both of q¹ and q² are H.

Further defining the embodiments of group A, B, C, D and E, a subgroupof any one of these embodiments is characterized by n being 1 and thesubstituent Y being a cationic substituent selected from NH₂, NHR, NR₂,NR₃ ⁺ and NHC(═NH)NH₂ (guanidyl), with R having the meaning outlinedabove. In some of these embodiments, the cationic substituent ispositioned on the ω-position (terminal C) of the alkyl chain beingfarthest away from the nucleoside ring (A¹ in embodiments of group A, A²in embodiments of group B, C, D and E).

EMBODIMENTS F

In some embodiments, the tricyclic nucleoside carries one substituentthat is defined by the following parameters: k is 0, h is 1, X_(h) is—O—, —C(═O)O— or —C(═O)NH— and A²-Y is (CH₂)_(m)CH₃, (CH₂)_(m)CH₂OH,(CH₂)_(m)CH₂NH₂, or (CH₂)_(m)CH₂NHC(═NH)NH₂, with m being 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In a subgroup thereof, thesubstituent as defined in the previous sentence is in position q³; inanother subgroup, the substituent as defined in the previous sentence isin position q⁴ or q⁵. All other positions of q³, q⁴ and q⁵ are H.Optionally, one of q¹ and q² is H and the other one is F or Cl, or bothof q¹ and q² are H.

Embodiments having a single substituent q³: In some embodiments, q¹, q²,q⁴ and q⁵ are H and q³ is a group described by the general formula-A¹-X_(h)-A²-Y_(n). Specific examples are given as groups G, H, I and J.

EMBODIMENTS G

In some embodiments, q³ is

-   -   —(CH₂)_(m)CH₃, —(CH₂)_(m)CH₂OH, (CH₂)_(m)CH₂NH₂,        (CH₂)_(m)CH₂NHC(═NH)NH₂, or    -   —CO(CH₂)_(m)CH₃, CO—(CH₂)_(m)CH₂OH, CO(CH₂)_(m)CH₂NH₂, or        CO(CH₂)_(m)CH₂NHC(═NH)NH₂,    -   —COO(CH₂)_(m)CH₃, COO(CH₂)_(m)CH₂OH, COO(CH₂)_(m)CH₂NH₂, or        COO(CH₂)_(m)CH₂NHC(═NH)NH₂, or    -   —CONH(CH₂)_(m)CH₃, CONH(CH₂)_(m)CH₂OH, CONH(CH₂)_(m)CH₂NH₂, or        CONH(CH₂)_(m)CH₂NHC(═NH)NH₂,    -   with m being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15        or 16. In a subgroup thereof, m is 3, 4, 5, 6, 7 or 8.

The length of the alkyl chain allows careful adjustment of delivery,bioavailability or permeability features of an oligonucleotide compoundinto which a nucleoside having this particular modification of q³ isincorporated.

EMBODIMENTS H

In some embodiments, q³ is a group described by the general formula-A¹-X_(h)-A²-Y_(n), wherein

-   -   k of A¹ is 0, h is 1, X_(h) is —O—, A² is C₅-alkyl and n of Y is        0, thus, q³ is —O—(CH₂)₄CH₃, or    -   k is 0, h is 1, X_(h) is —C(═O)O—, A² is C₆-alkyl and n is 0,        thus, q³ is —C(═O)O—(CH₂)₅CH₃, or    -   A¹ is C₂-alkyl, h is 1, X_(h) is —C(═O)O—, A² is C₆-alkyl and n        is 0, thus, q³ is —(CH₂)₂—C(═O)O—(CH₂)₅CH₃, or    -   k is 0, h is 1, X_(h) is —C(═O)—, A² is C₄-alkyl and n is 0,        thus, q³ is —C(═O)—(CH₂)₃CH₃, or    -   k is 0, h is 0, A² is C₈-alkyl, n is 1, and Y is —OH, wherein        the substituent group —OH is attached to the co-carbon atom of        the C₈-alkyl of A², thus, q³ is —(CH₂)₇CH₂OH, or    -   A¹ is —CH₂—, h is 1, X_(h) is —CO₂H, wherein i and n are each 0,        thus, q³ is —CH₂—COOH, or    -   A¹ is CH₂, h is 1, X_(h) is —C(═O)O—, A² is C₂-alkyl and n is 0,        thus, q³ is —CH₂—C(═O)O—CH₂CH₃, or    -   A¹ is CH₂, h is 1, X_(h) is —CONH₂, i and n are each 0, thus, q³        is —CH₂—CONH₂, or    -   A¹ is CH₂, h is 1, X_(h) is —C(═O)O—, A² is C₁₆-alkyl and n is        0, thus, q³ is CH₂—C(═O)O—(CH₂)₁₅CH₃, or    -   A¹ is CH₂, h is 1, X_(h) is —C(═O)O—, A² is C₃-alkyl, n is 1 and        Y_(n) is —NH₂, wherein the substituent group —NH₂ is attached to        the ω-carbon atom of the C₃-alkyl of A², thus, q³ is        CH₂—C(═O)O—(CH₂)₃NH₂, or    -   A¹ is CH₂, h is 1, X_(h) is —C(═O)NH—, A² is C₃-alkyl, n is 1        and Y_(n) is —NH₂, wherein the substituent group —NH₂ is        attached to the ω-carbon atom of the C₃-alkyl of A², thus, q³ is        CH₂—C(═O)NH—(CH₂)₃NH₂, or    -   A¹ is CH₂, h is 1, X_(h) is —C(═O)NH—, A² is C₃-alkyl, n is 1        and Y is —OH, wherein the substituent group —OH is attached to        the ω-carbon atom of the C₃-alkyl of A², thus, q³ is        CH₂—C(═O)NH—(CH₂)₃OH.

EMBODIMENTS I

In some embodiments, q³ is a group described by the general formula-A¹-X_(h)-A²-Y_(n), wherein A¹ is CH₂, h is 1, X_(h) is —C(═O)OR,C(═O)NR₂, —C(═O)O— or —C(═O)NR—, with each R being selectedindependently from H, methyl, ethyl, propyl, butyl, acetyl and2-hydroxyethyl in particular from H, methyl, ethyl, propyl and butyl. Ina subgroup thereof, z¹, z², q¹, q², q⁴ and q⁵ are H. In a furthersubgroup thereof, q³ is one of —CH₂COOH, —CH₂C(═O)OCH₂CH₃, —CH₂CONH₂,—CH₂C(═O)O(CH₂)₃NH₂, —CH₂COO(CH₂)₁₂₋₁₆CH₃, —CH₂COO(CH₂)₁₂₋₁₆NH₂, or—CH₂C(═O)O—(CH₂)₃NH(Fmoc).

EMBODIMENTS J

In some embodiments, q³ is one of —CH₂COOH, —CH₂C(═O)OCH₂CH₃, —CH₂CONH₂,—CH₂C(═O)O(CH₂)₃NH₂, —CH₂COO(CH₂)₁₂₋₁₆CH₃, or —CH₂COO(CH₂)₁₂₋₁₆NH₂, or—CH₂C(═O)O—(CH₂)₃NH(Fmoc).

In a further subgroup thereof, the tricyclic nucleoside is selected fromthe group of

-   -   a.

-   -   b.

-   -   c.

wherein Bx is selected from uracil, thymine, cytosine, 5-methylcytosine,adenine and guanine.

Also provided herein are nucleoside precursor compounds as exemplifiedby compound 8 of Example 1, compound 13 of Example 2 or compound 17 ofExample 3, in particular:

-   -   d.

-   -   e.

-   -   f.

wherein Bx is selected from uracil, thymine, cytosine, 5-methylcytosine,adenine and guanine.

EMBODIMENTS K

In some embodiments, k is 0, h is 1, X_(h) is —CR₂—, —C(═O)—, —C(═O)O—,—C(═O)NR—, —O—, —S—, —NR—, with each R being selected independently fromH, methyl, ethyl, propyl and butyl, A² is a C_(i)-alkyl, C_(i)-alkenylor C_(i)-alkynyl, with i being selected from any integer in the range of1 to 20, Y is a substituent group attached to any carbon atom on A²,selected from —F, —Cl, —Br, —OR, —SR, —NR₂, —CN, —NC, —NCO, —NCS, —SCN,—COR, —CO₂R, CONR₂, with each R being selected independently from H,methyl, ethyl, propyl, and butyl, and n is 0, 1, 2, 3, 4, 5 or 6.

EMBODIMENTS L

In some embodiments, k is 0, h is 1, X_(h) is —CR₂—, —C(═O)—, —C(═O)O—,—C(═O)NR—, —O—, —S—, —NR—, with each R being selected independently fromH, methyl, ethyl, propyl, and butyl, A² is a C_(i)-alkyl, C_(i)-alkenylor C_(i)-alkynyl, with i being selected from any integer in the range of1 to 20, Y is a substituent group attached to any carbon atom on A²,selected from —F, —Cl, —Br, —OR, —SR, —NR₂, —CN, —NC, —NCO, —NCS, —SCN,—COR, —CO₂R, CONR₂, with each R being selected independently from H,methyl, ethyl, propyl, and butyl, and n is 0, 1, 2, 3, 4, 5 or 6,wherein if z¹, z², q¹, q², q⁴ and q⁵ are H, q³ is one of —CH₂COOH,—CH₂C(═O)OCH₂CH₃, —CH₂CONH₂, —CH₂C(═O)O(CH₂)₃NH₂, —CH₂COO(CH₂)₁₂₋₁₆CH₃,—CH₂COO(CH₂)₁₂₋₁₆NH₂, or —CH₂C(═O)O—(CH₂)₃NH(Fmoc), in particular one of—CH₂C(═O)OCH₂CH₃, —CH₂C(═O)O(CH₂)₃NH₂, —CH₂COO(CH₂)₁₅CH₃ or—CH₂C(═O)O—(CH₂)₃NH(Fmoc).

EMBODIMENTS M

In some embodiments, A¹ is CH₂, h is 1, X_(h) is —CR₂—, —C(═O)—,—C(═O)O—, —C(═O)NR—, —O—, —S—, —NR—, with each R being selectedindependently from H, methyl, ethyl, propyl and butyl, A² is aC_(i)-alkyl, C_(i)-alkenyl or C_(i)-alkynyl, with i being selected fromany integer in the range of 1 to 20, Y is a substituent group attachedto any carbon atom on A², selected from —F, —Cl, —Br, —OR, —SR, —NR₂,—CN, —NC, —NCO, —NCS, —SCN, —COR, —CO₂R, CONR₂, with each R beingselected independently from H, methyl, ethyl, propyl, and butyl, and nis 0, 1, 2, 3, 4, 5 or 6.

EMBODIMENTS N

In some embodiments, A¹ is CH₂, h is 1, X_(h) is —CR₂—, —C(═O)—,—C(═O)O—, —C(═O)NR—, —O—, —S—, —NR—, with each R being selectedindependently from H, methyl, ethyl, propyl, and butyl, A² is aC_(i)-alkyl, C_(i)-alkenyl or C_(i)-alkynyl, with i being selected fromany integer in the range of 1 to 20, Y is a substituent group attachedto any carbon atom on A², selected from —F, —Cl, —Br, —OR, —SR, —NR₂,—CN, —NC, —NCO, —NCS, —SCN, —COR, —CO₂R, CONR₂, with each R beingselected independently from H, methyl, ethyl, propyl, and butyl, and nis 0, 1, 2, 3, 4, 5 or 6, wherein if z¹, z², q¹, q², q⁴ and q⁵ are H, q³is one of —CH₂COOH, —CH₂C(═O)OCH₂CH₃, —CH₂CONH₂, —CH₂C(═O)O(CH₂)₃NH₂,—CH₂COO(CH₂)₁₂₋₁₆CH₃, —CH₂COO(CH₂)₁₂₋₁₆NH₂, or—CH₂C(═O)O—(CH₂)₃NH(Fmoc), in particular one of —CH₂C(═O)OCH₂CH₃,—CH₂C(═O)O(CH₂)₃NH₂, —CH₂COO(CH₂)₁₅CH₃ or —CH₂C(═O)O—(CH₂)₃NH(Fmoc).

EMBODIMENTS O

In some embodiments, one of q³, q⁴ and q⁵ is selected from CH₂COOH,CH₂CONH₂, CH₂COO(CH₂)₃₋₆CH₃, —CH₂COO(CH₂)₃₋₇NH₂,—CH₂COO(CH₂)₃₋₇NHC(═NH)NH₂, CONH(CH₂)₃₋₆CH₃, CONH(CH₂)₃₋₇OH,CONH(CH₂)₃₋₇CH₂NH₂, or CONH(CH₂)₃₋₇CH₂NHC(═NH)NH₂.

In some embodiments, at least one of q₁, q₂, q₃, q₄ and q₅ is F, and oneof q₃, q₄ and q₅ is the group described by the general formula-A¹-X_(h)-A²-Y_(n).

In some embodiments, q³ and z¹ is F, and one of q⁴ and q⁵ is the groupdescribed by the general formula -A¹-X_(h)-A²-Y_(n).

In some embodiments, q³ is selected from the group described by thegeneral formula -A¹-X_(h)-A²-Y_(n) and one of z¹, z², q², q², q⁴ and q⁵is F.

In some embodiments, q³ is the group described by the general formula-A¹-X_(h)-A²-Y_(n), A¹ is CH₂, h is 1, X_(h) is —C(═O)OH, C(═O)NH₂—,—C(═O)O— or —C(═O)NR—, with each R being selected independently from H,methyl, ethyl, propyl, butyl, acetyl and 2-hydroxyethyl, in particularfrom H, methyl, ethyl, propyl, butyl, and one of z¹, z², q¹, q², q⁴ andq⁵ is F.

In some embodiments, two of q³, q⁴ and q⁵ are a group described by thegeneral formula -A¹-X_(h)-A²-Y_(n).

In some embodiments, q³ is a group described by the general formula-A¹-X_(h)-A²-Y_(n), wherein A¹ is CH₂, h is 1, X_(h) is —C(═O)OH,C(═O)NH—, —C(═O)O— or —C(═O)NR—, with each R being selectedindependently from H, methyl, ethyl, propyl, butyl, acetyl and2-hydroxyethyl, in particular from H, methyl, ethyl, propyl and butyl,and one of q⁴ or q⁵ is a group described by the general formula-A¹-X_(h)-A²-Y_(n) as defined above, while the other one is H.

According to a second aspect of the invention, an oligomeric compound isprovided comprising at least one tricyclic nucleoside having Formula II:

-   -   wherein independently for each tricyclic nucleoside of Formula        II:        -   Bx is a heterocyclic base moiety;        -   one of T³ and T⁴ is an internucleoside linking group            attaching the tricyclic nucleoside of Formula II to the            oligomeric compound and the other of T³ and T⁴ is hydroxyl,            a protected hydroxyl, a 5′ or 3′ terminal group or an            internucleoside linking group attaching the tricyclic            nucleoside to the oligomeric compound;        -   q¹ and q² are each, independently, H, F or Cl,        -   at least one of q³, q⁴ and q⁵ is, independently, a group            described by a general formula -A¹-X_(h)-A²-Y_(n), wherein        -   A¹ is a C_(k)-alkyl, C_(k)-alkenyl or C_(k)-alkynyl, with k            being an integer selected from the range of 0 to 20,        -   X_(h) is —C(═O)—, —C(═O)O—, —C(═O)NR—, —O—, —S—, —NR—,            —C(═O)R, —C(═O)OR, —C(═O)NR₂—, —OR, —SR or —NR₂, with each R            being selected independently from H, methyl, ethyl, propyl,            butyl, acetyl and 2-hydroxyethyl, and h is 0 or 1,        -   A² is a C_(i)-alkyl, C_(i)-alkenyl or C_(i)-alkynyl, with i            being an integer selected from the range of 0 to 20,        -   Y is a substituent group attached to any carbon atom on A¹            and/or A², selected from —F, —Cl, —Br, ═O, —OR, —SR, —NR₂,            —NR₃+, NHC(═NH)NH₂, —CN, —NC, —NCO, —NCS, —SCN, —COR, —CO₂R,            CONR₂. —R, with each R being selected independently from H,            methyl, ethyl, propyl, butyl, acetyl and 2-hydroxyethyl, and            n is 0, 1, 2, 3, 4, 5 or 6,        -   wherein k+i equals at least 1,    -   the other ones of q³, q⁴ and q⁵ are, independently, H, F or Cl,    -   one of z¹ and z² is H and the other of z¹ and z² is H, —OH, F,        Cl, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,        O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,        OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂.    -   and wherein said oligomeric compound comprises from 8 to 40        monomeric subunits.

In some embodiments of this aspect of the invention, Bx is a pyrimidine,substituted pyrimidine, purine or substituted purine. In someembodiments, Bx is selected from uracil, thymine, cytosine,5-methylcytosine, adenine and guanine. In some embodiments, Bx is anaromatic heterocyclic moiety capable of forming base pairs whenincorporated into DNA or RNA oligomers in lieu of the bases uracil,thymine, cytosine, 5-methylcytosine, adenine and guanine.

In some embodiments, one of z¹ and z² is F, OCH₃ or O(CH₂)₂—OCH₃ foreach tricyclic nucleoside of Formula II. In some embodiments, one of z¹and z² is F for each tricyclic nucleoside of Formula II. In someembodiments, z¹ and z² are each F for each tricyclic nucleoside ofFormula II. In some embodiments, one of z¹ and z² is F and the other oneis H for each tricyclic nucleoside of Formula II. In some embodiments,z¹ and z² are each H for each tricyclic nucleoside of Formula II.

In some embodiments, q¹ and q² are each H for each tricyclic nucleosideof Formula II. In some embodiments, one of q¹ and q² is F and the otheris H for each tricyclic nucleoside of Formula II. In some embodiments,q¹ and q² are each F for each tricyclic nucleoside of Formula II.

In some embodiments of this aspect of the invention, eachinternucleoside linking group is, independently, a phosphodiesterinternucleoside linking group or a phosphorothioate internucleosidelinking group. In some embodiments, essentially each internucleosidelinking group is a phosphorothioate internucleoside linking group.

In some embodiments, the oligomeric compound of the invention comprisesa first region having at least two contiguous tricyclic nucleosideshaving Formula II. In some embodiments, the oligomeric compound of theinvention comprises a first region having at least two contiguoustricyclic nucleosides having Formula II and a second region having atleast two contiguous monomeric subunits wherein each monomeric subunitin the second region is a modified nucleoside different from thetricyclic nucleosides of Formula II of said first region. According toanother alternative of this embodiment, the oligomeric compoundcomprises a third region located between said first and second regionswherein each monomer subunit in the third region is independently, anucleoside or a modified nucleoside that is different from eachtricyclic nucleoside of Formula II of the first region and each monomersubunit of the second region.

In some embodiments, the oligomeric compound of the invention comprisesa gapped oligomeric compound having an internal region of from 6 to 14contiguous monomer subunits flanked on each side by an external regionof from 1 to 5 contiguous monomer subunits wherein each monomer subunitin each external region is a tricyclic nucleoside of Formula II and eachmonomer subunit in the internal region is, independently, a nucleosideor modified nucleoside. In some embodiments, said internal regioncomprises from about 8 to about 14 contiguousβ-D-2′-deoxyribonucleosides. In some embodiments, said internal regioncomprises from about 9 to about 12 contiguousβ-D-2′-deoxyribonucleosides.

In some embodiments, each tricyclic nucleoside of Formula II comprisedin the oligomeric compound of the invention is selected from theembodiment group A above. In some embodiments, each tricyclic nucleosideof Formula II comprised in the oligomeric compound of the invention isselected from the embodiment group B above. In some embodiments, eachtricyclic nucleoside of Formula II comprised in the oligomeric compoundof the invention is selected from the embodiment group C above. In someembodiments, each tricyclic nucleoside of Formula II comprised in theoligomeric compound of the invention is selected from the embodimentgroup D above. In some embodiments, each tricyclic nucleoside of FormulaII comprised in the oligomeric compound of the invention is selectedfrom the embodiment group E above. In some embodiments, each tricyclicnucleoside of Formula II comprised in the oligomeric compound of theinvention is selected from the embodiment group F above. In someembodiments, each tricyclic nucleoside of Formula II comprised in theoligomeric compound of the invention is selected from the embodimentgroup G above. In some embodiments, each tricyclic nucleoside of FormulaII comprised in the oligomeric compound of the invention is selectedfrom the embodiment group H above. In some embodiments, each tricyclicnucleoside of Formula II comprised in the oligomeric compound of theinvention is selected from the embodiment group I above. In someembodiments, each tricyclic nucleoside of Formula II comprised in theoligomeric compound of the invention is selected from the embodimentgroup J above. In some embodiments, each tricyclic nucleoside of FormulaII comprised in the oligomeric compound of the invention is selectedfrom the embodiment group K above. In some embodiments, each tricyclicnucleoside of Formula II comprised in the oligomeric compound of theinvention is selected from the embodiment group L above. In someembodiments, each tricyclic nucleoside of Formula II comprised in theoligomeric compound of the invention is selected from the embodimentgroup M above. In some embodiments, each tricyclic nucleoside of FormulaII comprised in the oligomeric compound of the invention is selectedfrom the embodiment group N above.

In some embodiments, q³ is C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl,C₁-C₂₀ alkenyl, substituted C₁-C₂₀ alkenyl, C₁-C₂₀ alkynyl, substitutedC₁-C₂₀ alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino,substituted amino, thiol or substituted thiol for each tricyclicnucleoside of Formula II. In some embodiments, one of q⁴ and q⁵ is H andthe other of q⁴ and q⁵ is C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl, C₁-C₂₀alkenyl, substituted C₁-C₂₀ alkenyl, C₁-C₂₀ alkynyl, substituted C₁-C₂₀alkynyl, C₁-C₂₀ alkoxy, substituted C₁-C₂₀ alkoxy, amino, substitutedamino, thiol or substituted thiol for each tricyclic nucleoside ofFormula II.

In some embodiments, q³ is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆alkenyl, substituted C₁-C₆ alkenyl, C₁-C₆ alkynyl, substituted C₁-C₆alkynyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino, substitutedamino, thiol or substituted thiol for each tricyclic nucleoside ofFormula II. In some embodiments, one of q⁴ and q⁵ is H and the other ofq⁴ and q⁵ is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkenyl,substituted C₁-C₆ alkenyl, C₁-C₆ alkynyl, substituted C₁-C₆ alkynyl,C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino, substituted amino, thiolor substituted thiol for each tricyclic nucleoside of Formula II.

In some embodiments, the oligomeric compound of the invention comprisesone or several nucleotide blocks selected from the group consisting of

-   -   a.

-   -   b.

-   -   c.

-   -   -   wherein T³ and T⁴ have the meanings outlined above.

In some embodiments, at least two of q³, q⁴ and q⁵ are a group describedby the general formula formula -A¹-X_(h)-A²-Y_(n) for each tricyclicnucleoside of Formula II.

According to yet another aspect of the invention, a method forsolid-phase synthesis of an oligonucleotide is provided, comprising theuse of a trycyclic nucleoside according to the first aspect of theinvention. According to this aspect of the invention, a nucleosideaccording to the first aspect of the invention, any reactive OH, NH₂ orother reactive group being protected by a protective group as laid outelsewhere herein, is used e.g. as a phosphoamidite activated buildingblock and incorporated into the nascent oligomeric chain. The methodsand reagents useful for such purpose are known to the skilled person andare exemplified by the examples provided herein.

In certain embodiments, gapped oligomeric compounds are providedcomprising an internal region of from 6 to 14 contiguous monomersubunits flanked on each side by an external region of from 1 to 5contiguous monomer subunits wherein each monomer subunit in eachexternal region is a tricyclic nucleoside of Formula II and each monomersubunit in the internal region is, independently, a nucleoside ormodified nucleoside. In certain embodiments, the internal regioncomprises from about 8 to about 14 contiguousβ-D-2′-deoxyribonucleosides. In certain embodiments, the internal regioncomprises from about 9 to about 12 contiguousβ-D-2′-deoxyribonucleosides.

In certain embodiments, methods of inhibiting gene expression areprovided comprising contacting a cell with an oligomeric compoundcomprising a 5′ modified nucleoside as provided herein or a doublestranded composition comprising at least one oligomeric compoundcomprising a 5′ modified nucleoside as provided herein wherein saidoligomeric compound comprises from about 8 to about 40 monomericsubunits and is complementary to a target RNA. In certain embodiments,the cell is in an animal. In certain embodiments, the cell is in ahuman. In certain embodiments, the target RNA is selected from mRNA,pre-mRNA and micro RNA. In certain embodiments, the target RNA is mRNA.In certain embodiments, the target RNA is human mRNA. In certainembodiments, the target RNA is cleaved thereby inhibiting its function.In certain embodiments, the methods further comprise detecting thelevels of target RNA.

In certain embodiments, in vitro methods of inhibiting gene expressionare provided comprising contacting one or more cells or a tissue with anoligomeric compound or double stranded composition as provided herein.

In certain embodiments, oligomeric compounds or a double strandedcomposition as provided herein are used for use in an in vivo method ofinhibiting gene expression said method comprising contacting one or morecells, a tissue or an animal with one of the oligomeric compounds or adouble stranded composition as provided herein.

In certain embodiments, oligomeric compounds and double strandedcompositions as provided herein are used in medical therapy.

In certain embodiments, for each tricyclic nucleoside of Formula II, theplacement of the substituent group generally defined as-A¹-X_(h)-A²-Y_(n) at one of the substituent positions q³, q⁴, q⁵enhances biodistribution, cellular uptake or delivery of oligomers. Incertain embodiments, for each tricyclic nucleoside of Formula II, theplacement of the substituent group F at one of the substituent positionsq¹, q², q³, q⁴, q⁵, z¹ or z² enhances one or more properties of theoligomeric compound such as for example, and without limitation,stability, nuclease resistance, binding affinity, specificity,absorption, cellular distribution, cellular uptake, charge,pharmacodynamics and pharmacokinetics. In certain embodiments, for eachtricyclic nucleoside of Formula II, it is expected that the placement ofF at one of the substituent positions q¹, q², q³, q⁴, q⁵, z¹ or z² willenhance the binding affinity.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are novel tricyclic nucleosides having Formula I andoligomeric compounds prepared therefrom. The tricyclic nucleosideshaving Formula I are useful for enhancing one or more properties of theoligomeric compounds they are incorporated into such as but not limitedto nuclease resistance, cell entry, intracellular delivery, transport inthe body, particularly in the blood, ease of pharmaceutical formulationand drug metabolism. In certain embodiments, the oligomeric compoundsprovided herein hybridize to a portion of a target RNA resulting in lossof normal function of the target RNA. In certain embodiments, tricyclicnucleosides having Formula I are provided that can be incorporated intoantisense oligomeric compounds to reduce target RNA, such as messengerRNA, in vitro and in vivo. In one aspect the reduction or loss offunction of target RNA is useful for inhibition of gene expression vianumerous pathways. Such pathways include for example the steric blockingof transcription and/or translation of mRNA and cleavage of mRNA viasingle or double stranded oligomeric compounds. The oligomeric compoundsprovided herein are also expected to be useful as primers and probes indiagnostic applications.

In certain embodiments, double stranded compositions are providedwherein each double stranded composition comprises:

-   -   a first oligomeric compound and a second oligomeric compound        wherein the first oligomeric compound is complementary to the        second oligomeric compound and the second oligomeric compound is        complementary to a nucleic acid target;    -   at least one of the first and second oligomeric compounds        comprises at least one tricyclic nucleoside of Formula II; and    -   wherein said compositions optionally comprise one or more        terminal groups.

As used herein the term “alkyl,” refers to a saturated straight orbranched hydrocarbon radical containing up to 24, particularly up to 20,carbon atoms. Examples of alkyl groups include without limitation,methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyland the like. Alkyl groups typically include from 1 to about 20 carbonatoms, more typically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl)with from 1 to about 6 carbon atoms being more preferred. The term“lower alkyl” as used herein includes from 1 to about 6 carbon atoms.

As used herein the term “alkenyl,” refers to a straight or branchedhydrocarbon chain radical containing up to 24, particularly up to 20,carbon atoms and having at least one carbon-carbon double bond. Examplesof alkenyl groups include without limitation, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and thelike. Alkenyl groups typically include from 2 to about 20 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred.

As used herein the term “alkynyl,” refers to a straight or branchedhydrocarbon radical containing up to 24, particularly up to 20, carbonatoms and having at least one carbon-carbon triple bond. Examples ofalkynyl groups include, without limitation, ethynyl, 1-propynyl,1-butynyl, and the like. Alkynyl groups typically include from 2 toabout 20 carbon atoms, more typically from 2 to about 12 carbon atomswith from 2 to about 6 carbon atoms being more preferred.

As used herein the term “alkoxy,” refers to a radical formed between analkyl group and an oxygen atom wherein the oxygen atom is used to attachthe alkoxy group to a parent molecule. Examples of alkoxy groups includewithout limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.Alkoxy groups as used herein may optionally include further substituentgroups.

As used herein the term “aminoalkyl” refers to an amino substitutedC₁-C₁₂ alkyl radical. The alkyl portion of the radical forms a covalentbond with a parent molecule. The amino group can be located at anyposition and the aminoalkyl group can be substituted with a furthersubstituent group at the alkyl and/or amino portions.

As used herein the term “protecting group,” refers to a labile chemicalmoiety, which is known in the art to protect reactive groups includingwithout limitation, hydroxyl, amino and thiol groups, against undesiredreactions during synthetic procedures. Protecting groups are typicallyused selectively to protect sites during reactions at other reactivesites and can then be removed to leave the unprotected group as is oravailable for further reactions. Protecting groups as known in the artare described generally in Greene's Protective Groups in OrganicSynthesis, 4th edition, John Wiley & Sons, New York, 2007.

Groups can be selectively incorporated into oligomeric compounds asprovided herein as precursors. For example an amino group can be placedinto a compound as provided herein as an azido group that can bechemically converted to the amino group at a desired point in thesynthesis. Generally, groups are protected or present as precursors thatwill be inert to reactions that modify other areas of the parentmolecule for conversion into their final groups at an appropriate time.Further representative protecting or precursor groups are discussed inAgrawal et al., Protocols for Oligonucleotide Conjugates, Humana Press;New Jersey, 1994, 26, 1-72.

Examples of hydroxyl protecting groups include without limitation,acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxyl)ethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl(TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl,pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl,dimethoxytrityl (DMT), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Wherein morecommonly used hydroxyl protecting groups include without limitation,benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyl-diphenylsilyl,benzoyl, mesylate, tosylate, dimethoxytrityl (DMT),9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl(MOX).

Examples of amino protecting groups include without limitation,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; andimine- and cyclic imide-protecting groups, such as phthalimido anddithiasuccinoyl.

Examples of thiol protecting groups include without limitation,triphenylmethyl (trityl), benzyl (Bn), and the like.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R) or (S). Included herein are all such possibleisomers, as well as their racemic and optically pure forms. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to limit a particularconfiguration unless the text so states.

In some embodiments, an alkyl, alkenyl or alkynyl group as used hereinmay optionally include one or more further substituent groups. The terms“substituent” and “substituent group” are meant to include groups thatare typically added to other groups or parent compounds to enhancedesired properties or provide other desired effects. Substituent groupscan be protected or unprotected and can be added to one available siteor to many available sites in a parent compound. Substituent groups mayalso be further substituted with other substituent groups and may beattached directly or via a linking group such as an alkyl or hydrocarbylgroup to a parent compound.

Substituent groups amenable herein include without limitation, halogen,oxygen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl(—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substitutedoxy (—O—R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl,heteroarylalkyl, amino (—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido(—C(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro(—NO₂), cyano (—CN), isocyano (—NC), cyanato (—OCN), isocyanato (—NCO),thiocyanato (—SCN); isothiocyanato (—NCS); carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))(R,)), guanidinyl(—N(R_(bb))C(═NR_(bb))—N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cc) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, H, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

In this context, “recursive substituent” means that a substituent mayrecite another instance of itself. Because of the recursive nature ofsuch substituents, theoretically, a large number may be present in anygiven claim. One of ordinary skill in the art of medicinal chemistry andorganic chemistry understands that the total number of such substituentsis reasonably limited by the desired properties of the compoundintended. Such properties include, by way of example and not limitation,physical properties such as molecular weight, solubility or log P,application properties such as activity against the intended target andpractical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above.

In some embodiments, an alkyl, alkenyl or alkynyl group as used hereincontains one, two or three further substituent groups selectedindependently from the group of —F, —Cl, —Br, ═O, NH₂, SH, OH, OR, SR,NHR, —NR₂, —CN, —NC, —NCO, —NCS, —SCN, —COR, —CO₂R, CONR₂, —R with Rbeing selected from methyl, ethyl, propyl, butyl, acetyl and2-hydroxyethyl.

In some embodiments, an alkyl, alkenyl or alkynyl group as used hereincontains no further substituent groups but consists only of carbon andhydrogen atoms.

As used herein, the term “nucleobase” refers to unmodified or naturallyoccurring nucleobases which include, but are not limited to, the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). As used herein, the term “heterocyclic basemoiety” refers to unmodified or naturally occurring nucleobases as wellas modified or non-naturally occurring nucleobases and syntheticmimetics thereof (such as for example phenoxazines). In certainembodiments, a heterocyclic base moiety is any heterocyclic system thatcontains one or more atoms or groups of atoms capable of hydrogenbonding to a heterocyclic base of a nucleic acid.

In certain embodiments, heterocyclic base moieties include withoutlimitation modified nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-amino-adenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein.

In certain embodiments, heterocyclic base moieties include withoutlimitation tricyclic pyrimidines such as 1,3-diazaphenoxazine-2-one,1,3-diazaphenothiazine-2-one and9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Heterocyclicbase moieties also include those in which the purine or pyrimidine baseis replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further heterocyclicbase moieties include without limitation those known to the art skilled(see for example: U.S. Pat. No. 3,687,808; Swayze et al., The MedicinalChemistry of Oligonucleotides in Antisense a Drug Technology, Chapter 6,pages 143-182, Crooke, S. T., ed., 2008); The Concise Encyclopedia OfPolymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley &Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, InternationalEdition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Researchand Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993,273-302.

As used herein the term “sugar moiety” refers to naturally occurringsugars having a furanose ring, synthetic or non-naturally occurringsugars having a modified furanose ring and sugar surrogates wherein thefuranose ring has been replaced with a cyclic ring system such as forexample a morpholino or hexitol ring system or a non-cyclic sugarsurrogate such as that used in peptide nucleic acids. Illustrativeexamples of sugar moieties useful in the preparation of oligomericcompounds include without limitation, β-D-ribose, β-D-2′-deoxyribose,substituted sugars (such as 2′, 5′ and bis substituted sugars),4′-S-sugars (such as 4′-S-ribose, 4′-S-2′-deoxyribose and4′-S-2′-substituted ribose), tricyclic modified sugars (such as the2′-O—CH₂-4′ or 2′-O—(CH₂)₂-4′ bridged ribose derived tricyclic sugars)and sugar surrogates (such as for example when the ribose ring has beenreplaced with a morpholino, a hexitol ring system or an open non-cyclicsystem).

As used herein, the term “nucleoside” refers to a nucleobase-sugarcombination. The two most common classes of such nucleobases are purinesand pyrimidines.

As used herein, the term nucleotide refers to a nucleoside furthercomprising a modified or unmodified phosphate internucleoside linkinggroup or a non-phosphate internucleoside linking group. For nucleotidesthat include a pentofuranosyl sugar, the internucleoside linking groupcan be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar.The phosphate and or a non-phosphate internucleoside linking groups areroutinely used to covalently link adjacent nucleosides to one another toform a linear polymeric compound.

The term “nucleotide mimetic” as used herein is meant to includemonomers that incorporate into oligomeric compounds with sugar andlinkage surrogate groups, such as for example peptide nucleic acids(PNA) or morpholinos (linked by —N(H)—C(═O)—O—). In general, theheterocyclic base at each position is maintained for hybridization to anucleic acid target but the sugar and linkage is replaced with surrogategroups that are expected to function similar to native groups but haveone or more enhanced properties.

As used herein the term “nucleoside mimetic” is intended to includethose structures used to replace the sugar and the base at one or morepositions of an oligomeric compound. Examples of nucleoside mimeticsinclude without limitation nucleosides wherein the heterocyclic basemoiety is replaced with a phenoxazine moiety (for example the9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one group, also referred to asa G-clamp which forms four hydrogen bonds when hybridized with aguanosine base) and further replacement of the sugar moiety with a groupsuch as for example a morpholino, a cyclohexenyl or abicyclo[3.1.0]hexyl.

As used herein the term “modified nucleoside” is meant to include allmanner of modified nucleosides that can be incorporated into anoligomeric compound using oligomer synthesis. The term is intended toinclude modifications made to a nucleoside such as modifiedstereochemical configurations, one or more substitutions, and deletionof groups as opposed to the use of surrogate groups which are describedelsewhere herein. The term includes nucleosides having a furanose sugar(or 4′-S analog) portion and can include a heterocyclic base or can bean abasic nucleoside. One group of representative modified nucleosidesincludes without limitation, substituted nucleosides (such as 2′, 5′,and/or 4′ substituted nucleosides) 4′-S-modified nucleosides, (such as4′-S-ribonucleosides, 4′-S-2′-deoxyribonucleosides and4′-S-2′-substituted ribonucleosides), bicyclic modified nucleosides(such as for example, bicyclic nucleosides wherein the sugar moiety hasa 2′-O—CHR_(a)-4′ bridging group, wherein R_(a) is H, alkyl orsubstituted alkyl) and base modified nucleosides. The sugar can bemodified with more than one of these modifications listed such as forexample a bicyclic modified nucleoside further including a5′-substitution or a 5′ or 4′ substituted nucleoside further including a2′ substituent. The term modified nucleoside also includes combinationsof these modifications such as base and sugar modified nucleosides.These modifications are meant to be illustrative and not exhaustive asother modifications are known in the art and are also envisioned aspossible modifications for the modified nucleosides described herein. Asused herein the term “monomer subunit” is meant to include all manner ofmonomer units that are amenable to oligomer synthesis with one preferredlist including monomer subunits such as β-D-ribonucleosides,β-D-2′-deoxyribnucleosides, modified nucleosides, including substitutednucleosides (such as 2′, 5′ and bis substituted nucleosides),4′-S-modified nucleosides, (such as 4′-S-ribonucleosides,4′-S-2′-deoxyribonucleosides and 4′-S-2′-substituted ribonucleosides),bicyclic modified nucleosides (such as bicyclic nucleosides wherein thesugar moiety has a 2′-O—CHR_(a)-4′ bridging group, wherein R_(a) is H,alkyl or substituted alkyl), other modified nucleosides, nucleosidemimetics, nucleosides having sugar surrogates and the tricyclicnucleosides as provided herein. Many other monocyclic, bicyclic andtricyclic ring systems are known in the art and are suitable as sugarsurrogates that can be used to modify nucleosides for incorporation intooligomeric compounds as provided herein (see for example review article:Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854). Suchring systems can undergo various additional substitutions to furtherenhance their activity.

As used herein the term “reactive phosphorus” is meant to include groupsthat are covalently linked to a monomer subunit that can be furtherattached to an oligomeric compound that are useful for forminginternucleoside linkages including for example phosphodiester andphosphorothioate internucleoside linkages. Such reactive phosphorusgroups are known in the art and contain phosphorus atoms in P^(III) orP^(V) valence state including, but not limited to, phosphoramidite,H-phosphonate, phosphate triesters and phosphorus containing chiralauxiliaries. In certain embodiments, reactive phosphorus groups areselected from diisopropylcyanoethoxy phosphoramidite(—O*—P[N[(CH(CH₃)₂]₂]O(CH₂)₂CN) and H-phosphonate (—O*—P(═O)(H)OH),wherein the O* is provided from the Markush group for the monomer. Apreferred synthetic solid phase synthesis utilizes phosphoramidites(P^(III) chemistry) as reactive phosphites. The intermediate phosphitecompounds are subsequently oxidized to the phosphate or thiophosphate(P^(V) chemistry) using known methods to yield, phosphodiester orphosphorothioate internucleoside linkages. Additional reactivephosphates and phosphites are disclosed in Tetrahedron Report Number 309(Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

The term “oligonucleoside” refers to a sequence of nucleosides that arejoined by internucleoside linkages that do not have phosphorus atoms.Internucleoside linkages of this type include short chain alkyl,cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one ormore short chain heteroatomic and one or more short chain heterocyclic.These internucleoside linkages include without limitation, siloxane,sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl,methylene formacetyl, thioformacetyl, alkeneyl, sulfamate,methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide andothers having mixed N, O, S and CH₂ component parts.

As used herein, the term “oligomeric compound” refers to a contiguoussequence of linked monomer subunits. Each linked monomer subunitnormally includes a heterocyclic base moiety but monomer subunits alsoincludes those without a heterocyclic base moiety such as abasic monomersubunits. At least some and generally most if not essentially all of theheterocyclic bases in an oligomeric compound are capable of hybridizingto a nucleic acid molecule, normally a preselected RNA target. The term“oligomeric compound” therefore includes oligonucleotides,oligonucleotide analogs and oligonucleosides. It also includes polymershaving one or a plurality of nucleoside mimetics and or nucleosideshaving sugar surrogate groups.

In certain embodiments, oligomeric compounds comprise a plurality ofmonomer subunits independently selected from naturally occurringnucleosides, non-naturally occurring nucleosides, modified nucleosides,nucleoside mimetics, and nucleosides having sugar surrogate groups. Incertain embodiments, oligomeric compounds are single stranded. Incertain embodiments, oligomeric compounds are double stranded comprisinga double-stranded duplex. In certain embodiments, oligomeric compoundscomprise one or more conjugate groups and/or terminal groups.

As used herein the term “internucleoside linkage” or “internucleosidelinking group” is meant to include all manner of internucleoside linkinggroups known in the art including but not limited to, phosphoruscontaining internucleoside linking groups such as phosphodiester andphosphorothioate, and non-phosphorus containing internucleoside linkinggroups such as formacetyl and methyleneimino. Internucleoside linkagesalso includes neutral non-ionic internucleoside linkages such as amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′) andmethylphosphonate wherein a phosphorus atom is not always present.

In certain embodiments, oligomeric compounds as provided herein can beprepared having one or more internucleoside linkages containing modifiede.g. non-naturally occurring internucleoside linkages. The two mainclasses of internucleoside linkages are defined by the presence orabsence of a phosphorus atom. Modified internucleoside linkages having aphosphorus atom include without limitation, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity can comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

In certain embodiments, oligomeric compounds as provided herein can beprepared having one or more non-phosphorus containing internucleosidelinkages. Such oligomeric compounds include without limitation, thosethat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include those having siloxane backbones;sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include without limitation, U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,677,439;5,646,269 and 5,792,608, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

In certain embodiments, the oligomeric compounds as provided herein canbe modified by covalent attachment of one or more conjugate groups. Ingeneral, conjugate groups modify one or more properties of theoligomeric compounds they are attached to. Such oligonucleotideproperties include without limitation, pharmacodynamics,pharmacokinetics, binding, absorption, cellular distribution, cellularuptake, charge and clearance. Conjugate groups are routinely used in thechemical arts and are linked directly or via an optional linking moietyor linking group to a parent compound such as an oligomeric compound. Apreferred list of conjugate groups includes without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes.

In certain embodiments, the oligomeric compounds as provided herein canbe modified by covalent attachment of one or more terminal groups to the5′ or 3′-terminal groups. A terminal group can also be attached at anyother position at one of the terminal ends of the oligomeric compound.As used herein the terms “5′-terminal group”, “3′-terminal group”,“terminal group” and combinations thereof are meant to include usefulgroups known to the art skilled that can be placed on one or both of theterminal ends, including but not limited to the 5′ and 3′-ends of anoligomeric compound respectively, for various purposes such as enablingthe tracking of the oligomeric compound (a fluorescent label or otherreporter group), improving the pharmacokinetics or pharmacodynamics ofthe oligomeric compound (such as for example: uptake and/or delivery) orenhancing one or more other desirable properties of the oligomericcompound (a group for improving nuclease stability or binding affinity).In certain embodiments, 5′ and 3′-terminal groups include withoutlimitation, modified or unmodified nucleosides; two or more linkednucleosides that are independently, modified or unmodified; conjugategroups; capping groups; phosphate moieties; and protecting groups.

As used herein the term “phosphate moiety” refers to a terminalphosphate group that includes phosphates as well as modified phosphates.The phosphate moiety can be located at either terminus but is preferredat the 5′-terminal nucleoside. In one aspect, the terminal phosphate isunmodified having the formula —O—P(═O)(OH)OH. In another aspect, theterminal phosphate is modified such that one or more of the O and OHgroups are replaced with H, O, S, N(R) or alkyl where R is H, an aminoprotecting group or unsubstituted or substituted alkyl. In certainembodiments, the 5′ and or 3′ terminal group can comprise from 1 to 3phosphate moieties that are each, independently, unmodified (di ortri-phosphates) or modified.

As used herein, the term “phosphorus moiety” refers to a group havingthe formula:

wherein:

R_(x) and R_(y) are each, independently, hydroxyl, protected hydroxylgroup, thiol, protected thiol group, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, a protected amino orsubstituted amino; and

R_(z) is O or S.

As a monomer such as a phosphoramidite or H-phosphonate the protectedphosphorus moiety is preferred to maintain stability during oligomersynthesis. After incorporation into an oligomeric compound thephosphorus moiety can include deprotected groups.

Phosphorus moieties included herein can be attached to a monomer, whichcan be used in the preparation of oligomeric compounds, wherein themonomer may be attached using O, S, NR_(d) or CR_(e)R_(f), wherein R_(d)includes without limitation H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl,C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or substituted acyl,and R_(e) and R_(f) each, independently, include without limitation H,halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy orsubstituted C₁-C₆ alkoxy. Such linked phosphorus moieties includewithout limitation, phosphates, modified phosphates, thiophosphates,modified thiophosphates, phosphonates, modified phosphonates,phosphoramidates and modified phosphoramidates.

The relative ability of a chemically-modified oligomeric compound tobind to complementary nucleic acid strands, as compared to naturaloligonucleotides, is measured by obtaining the melting temperature of ahybridization complex of said chemically-modified oligomeric compoundwith its complementary unmodified target nucleic acid. The meltingtemperature (T_(m)), a characteristic physical property of doublehelixes, denotes the temperature in degrees centigrade at which 50%helical versus coiled (unhybridized) forms are present. T_(m) (alsocommonly referred to as binding affinity) is measured by using the UVspectrum to determine the formation and breakdown (melting) ofhybridization. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently a reduction in UV absorption indicates a higher T_(m).

It is known in the art that the relative duplex stability of anantisense compound:RNA target duplex can be modulated throughincorporation of chemically-modified nucleosides into the antisensecompound. Sugar-modified nucleosides have provided the most efficientmeans of modulating the T_(m) of an antisense compound with its targetRNA. Sugar-modified nucleosides that increase the population of or lockthe sugar in the C3′-endo (Northern, RNA-like sugar pucker)configuration have predominantly provided a per modification T_(m)increase for antisense compounds toward a complementary RNA target.Sugar-modified nucleosides that increase the population of or lock thesugar in the C2′-endo (Southern, DNA-like sugar pucker) configurationpredominantly provide a per modification Tm decrease for antisensecompounds toward a complementary RNA target. The sugar pucker of a givensugar-modified nucleoside is not the only factor that dictates theability of the nucleoside to increase or decrease an antisensecompound's T_(m) toward complementary RNA. For example, thesugar-modified nucleoside tricycloDNA is predominantly in the C2′-endoconformation, however it imparts a 1.9 to 3° C. per modificationincrease in T_(m) toward a complementary RNA. Another example of asugar-modified high-affinity nucleoside that does not adopt the C3′-endoconformation is α-L-LNA (described in more detail herein).

As used herein, “T_(m)” (melting temperature) is the temperature atwhich the two strands of a duplex nucleic acid separate. The T_(m) isoften used as a measure of duplex stability of an antisense compoundtoward a complementary RNA molecule.

As used herein, “complementarity” in reference to nucleobases refers toa nucleobase that is capable of base pairing with another nucleobase.For example, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.Nucleobases or more broadly, heterocyclic base moieties, comprisingcertain modifications may maintain the ability to pair with acounterpart nucleobase and thus, are still capable of complementarity.

As used herein, “non-complementary” in reference to nucleobases refersto a pair of nucleobases that do not form hydrogen bonds with oneanother or otherwise support hybridization.

As used herein, “complementary” in reference to linked nucleosides,oligonucleotides, oligomeric compounds, or nucleic acids, refers to thecapacity of an oligomeric compound to hybridize to another oligomericcompound or nucleic acid through nucleobase or more broadly,heterocyclic base, complementarity. In certain embodiments, an antisensecompound and its target are complementary to each other when asufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%complementary to a target nucleic acid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomeric compound mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). In certain embodiments, oligomericcompounds can comprise at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or at least about 99% sequencecomplementarity to a target region within the target nucleic acidsequence to which they are targeted. For example, an oligomeric compoundin which 18 of 20 nucleobases of the oligomeric compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an oligomericcompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin this scope. Percent complementarity of an oligomeric compoundwith a region of a target nucleic acid can be determined routinely usingBLAST programs (basic local alignment search tools) and PowerBLASTprograms known in the art (Altschul et al., J. Mol. Biol., 1990, 215,403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

As used herein, “hybridization” refers to the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymidine and uracil which pair through the formation ofhydrogen bonds. The natural base guanine is nucleobase complementary tothe natural bases cytosine and 5-methyl cytosine. Hybridization canoccur under varying circumstances.

As used herein, “target nucleic acid” refers to any nucleic acidmolecule the expression, amount, or activity of which is capable ofbeing modulated by an antisense compound. In certain embodiments, thetarget nucleic acid is DNA or RNA. In certain embodiments, the targetRNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA,mature microRNA, promoter-directed RNA, or natural antisensetranscripts. For example, the target nucleic acid can be a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In certain embodiments, target nucleic acid is aviral or bacterial nucleic acid.

Further included herein are oligomeric compounds such as antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these oligomeric compoundsmay be introduced in the form of single-stranded, double-stranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges or loops. Once introducedto a system, the oligomeric compounds provided herein may elicit theaction of one or more enzymes or structural proteins to effectmodification of the target nucleic acid. Alternatively, the oligomericcompound may inhibit the activity the target nucleic acid through anoccupancy-based method, thus interfering with the activity of the targetnucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded oligomeric compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While one form of oligomeric compound is a single-stranded antisenseoligonucleotide, in many species the introduction of double-strandedstructures, such as double-stranded RNA (dsRNA) molecules, has beenshown to induce potent and specific antisense-mediated reduction of thefunction of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the desired activity of the compound and do not impartundesired toxicological effects thereto. The term “pharmaceuticallyacceptable salt” includes a salt prepared from pharmaceuticallyacceptable non-toxic acids or bases, including inorganic or organicacids and bases.

Pharmaceutically acceptable salts of the oligomeric compounds describedherein may be prepared by methods well-known in the art. For a review ofpharmaceutically acceptable salts, see Stahl and Wermuth, Handbook ofPharmaceutical Salts: Properties, Selection and Use (Wiley-VCH,Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides areuseful and are well accepted for therapeutic administration to humans.Accordingly, in certain embodiments the oligomeric compounds describedherein are in the form of a sodium salt.

In certain embodiments, oligomeric compounds provided herein comprisefrom about 8 to about 80 monomer subunits in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,or 80 monomer subunits in length, or any range therewithin.

Oligomeric compounds are routinely prepared using solid support methodsas opposed to solution phase methods. Commercially available equipmentcommonly used for the preparation of oligomeric compounds that utilizethe solid support method is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed. Suitable solid phase techniques, including automated synthesistechniques, are described in Oligonucleotides and Analogues, a PracticalApproach, F. Eckstein, Ed., Oxford University Press, New York, 1991.

The synthesis of RNA and related analogs relative to the synthesis ofDNA and related analogs has been increasing as efforts in RNAinterference and micro RNA increase. The primary RNA synthesisstrategies that are presently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS),5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),2′-O-[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM) and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. The primary groups being used for commercial RNA synthesisare: TBDMS: 5′-O-DMT-2′-O-t-butyldimethylsilyl; TOM:2′-O-[(triisopropylsilyl)oxy]methyl; DOD/ACE:(5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether-2′-O-bis(2-acetoxyethoxy)methyl; and FPMP:5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-ethoxypiperidin-4-yl]. In certainembodiments, each of the aforementioned RNA synthesis strategies can beused herein. In certain embodiments, the aforementioned RNA synthesisstrategies can be performed together in a hybrid fashion e.g. using a5′-protecting group from one strategy with a 2′-O-protecting fromanother strategy.

In some embodiments, “suitable target segments” may be employed in ascreen for additional oligomeric compounds that modulate the expressionof a selected protein. “Modulators” are those oligomeric compounds thatdecrease or increase the expression of a nucleic acid molecule encodinga protein and which comprise at least an 8-nucleobase portion which iscomplementary to a suitable target segment. The screening methodcomprises the steps of contacting a suitable target segment of a nucleicacid molecule encoding a protein with one or more candidate modulators,and selecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encoding a protein.Once it is shown that the candidate modulator or modulators are capableof modulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding a peptide, the modulator may then beemployed herein in further investigative studies of the function of thepeptide, or for use as a research, diagnostic, or therapeutic agent. Inthe case of oligomeric compounds targeted to microRNA, candidatemodulators may be evaluated by the extent to which they increase theexpression of a microRNA target RNA or protein (as interference with theactivity of a microRNA will result in the increased expression of one ormore targets of the microRNA).

As used herein, “expression” refers to the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, splicing, post-transcriptional modification, andtranslation.

Suitable target segments may also be combined with their respectivecomplementary oligomeric compounds provided herein to form stabilizeddouble-stranded (duplexed) oligonucleotides. Such double strandedoligonucleotide moieties have been shown in the art to modulate targetexpression and regulate translation as well as RNA processing via anantisense mechanism. Moreover, the double-stranded moieties may besubject to chemical modifications (Fire et al., Nature, 1998, 391,806-811; Timmons and Fire, Nature, 1998, 395, 854; Timmons et al., Gene,2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431;Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507;Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,2001, 411, 494-498; Elbashir et al., Genes Dev., 2001, 15, 188-200). Forexample, such double-stranded moieties have been shown to inhibit thetarget by the classical hybridization of antisense strand of the duplexto the target, thereby triggering enzymatic degradation of the target(Tijsterman et al., Science, 2002, 295, 694-697).

The oligomeric compounds provided herein can also be applied in theareas of drug discovery and target validation. In certain embodiments,provided herein is the use of the oligomeric compounds and targetsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between proteins and a disease state, phenotype, orcondition. These methods include detecting or modulating a targetpeptide comprising contacting a sample, tissue, cell, or organism withone or more oligomeric compounds provided herein, measuring the nucleicacid or protein level of the target and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther oligomeric compound as provided herein. These methods can alsobe performed in parallel or in combination with other experiments todetermine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a particular disease, condition,or phenotype. In certain embodiments, oligomeric compounds are providedfor use in therapy. In certain embodiments, the therapy is reducingtarget messenger RNA.

As used herein, the term “dose” refers to a specified quantity of apharmaceutical agent provided in a single administration. In certainembodiments, a dose may be administered in two or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired, the desired dose requires a volume not easilyaccommodated by a single injection. In such embodiments, two or moreinjections may be used to achieve the desired dose. In certainembodiments, a dose may be administered in two or more injections tominimize injection site reaction in an individual.

In certain embodiments, chemically-modified oligomeric compounds areprovided herein that may have a higher affinity for target RNAs thandoes non-modified DNA. In certain such embodiments, higher affinity inturn provides increased potency allowing for the administration of lowerdoses of such compounds, reduced potential for toxicity, improvement intherapeutic index and decreased overall cost of therapy.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature, 2001, 411,494-498; Nishikura et al., Cell, 2001, 107, 415-416; and Bass et al.,Cell, 2000, 101, 235-238.)

In certain embodiments, oligomeric compounds provided herein can beutilized for diagnostics, therapeutics, prophylaxis and as researchreagents and kits. Furthermore, antisense oligonucleotides, which areable to inhibit gene expression with exquisite specificity, are oftenused by those of ordinary skill to elucidate the function of particulargenes or to distinguish between functions of various members of abiological pathway. In certain embodiments, oligomeric compoundsprovided herein can be utilized either alone or in combination withother oligomeric compounds or other therapeutics as tools indifferential and/or combinatorial analyses to elucidate expressionpatterns of a portion or the entire complement of genes expressed withincells and tissues. Oligomeric compounds can also be effectively used asprimers and probes under conditions favoring gene amplification ordetection, respectively. These primers and probes are useful in methodsrequiring the specific detection of nucleic acid molecules encodingproteins and in the amplification of the nucleic acid molecules fordetection or for use in further studies. Hybridization of oligomericcompounds as provided herein, particularly the primers and probes, witha nucleic acid can be detected by means known in the art. Such means mayinclude conjugation of an enzyme to the oligonucleotide, radiolabellingof the oligonucleotide or any other suitable detection means. Kits usingsuch detection means for detecting the level of selected proteins in asample may also be prepared.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more of the oligomeric compounds provided herein arecompared to control cells or tissues not treated with oligomericcompounds and the patterns produced are analyzed for differential levelsof gene expression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds and or oligomeric compounds which affectexpression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.USA, 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

Those skilled in the art, having possession of the present disclosurewill be able to prepare oligomeric compounds, comprising a contiguoussequence of linked monomer subunits, of essentially any viable length topractice the methods disclosed herein. Such oligomeric compounds willinclude at least one and preferably a plurality of the tricyclicnucleosides provided herein and may also include other monomer subunitsincluding but not limited to nucleosides, modified nucleosides,nucleosides comprising sugar surrogate groups and nucleoside mimetics.

While in certain embodiments, oligomeric compounds provided herein canbe utilized as described, the following examples serve only toillustrate and are not intended to be limiting. Wherever alternativesfor single separable features such as, for example, any of thealternatives given for q¹, q⁴, T¹, or T², or Bx, or A¹, A², X, h, Y orn, are laid out herein as “embodiments”, it is to be understood thatsuch alternatives may be combined freely to form discrete embodiments ofthe invention disclosed herein.

EXAMPLES General Methods

¹H and ¹³C NMR spectra were recorded on a 300 MHz and 75 MHz Brukerspectrometer, respectively.

Synthesis of Nucleoside Phosphoramidites

The preparation of nucleoside phosphoramidites is performed followingprocedures that are illustrated herein and in the art such as but notlimited to U.S. Pat. No. 6,426,220 and WO02/36743.

Synthesis of Oligomeric Compounds

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through solid phase synthesis.

Oligomeric compounds: Unsubstituted and substituted phosphodiester (P═O)oligomeric compounds can be synthesized on an automated DNA synthesizer(for example Applied Biosystems model 394) using standardphosphoramidite chemistry with oxidation by iodine.

In certain embodiments, phosphorothioate internucleoside linkages (P═S)are synthesized similar to phosphodiester internucleoside linkages withthe following exceptions: thiation is effected by utilizing a 10% w/vsolution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forthe oxidation of the phosphite linkages. The thiation reaction step timeis increased to 180 sec and preceded by the normal capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (12-16 hr), the oligomeric compounds are recoveredby precipitating with greater than 3 volumes of ethanol from a 1 MNH₄OAc solution. Phosphinate internucleoside linkages can be prepared asdescribed in U.S. Pat. No. 5,508,270. Alkyl phosphonate internucleosidelinkages can be prepared as described in U.S. Pat. No. 4,469,863.3′-Deoxy-3′-methylene phosphonate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.Phosphoramidite internucleoside linkages can be prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.Alkylphosphonothioate internucleoside linkages can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).3′-Deoxy-3′-amino phosphoramidate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,476,925. Phosphotriesterinternucleoside linkages can be prepared as described in U.S. Pat. No.5,023,243. Borano phosphate internucleoside linkages can be prepared asdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. Oligomericcompounds having one or more non-phosphorus containing internucleosidelinkages including without limitation methylenemethylimino linkedoligonucleosides, also identified as MMI linked oligonucleosides,methylenedimethylhydrazo linked oligonucleosides, also identified as MDHlinked oligonucleosides, methylenecarbonylamino linked oligonucleosides,also identified as amide-3 linked oligonucleosides, andmethyleneaminocarbonyl linked oligonucleosides, also identified asamide-4 linked oligonucleosides, as well as mixed backbone oligomericcompounds having, for instance, alternating MMI and P═O or P═S linkagescan be prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal internucleoside linkages can be preparedas described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxideinternucleoside linkages can be prepared as described in U.S. Pat. No.5,223,618.

Isolation and Purification of Oligomeric Compounds

After cleavage from the controlled pore glass solid support or othersupport medium and deblocking in concentrated ammonium hydroxide at 55°C. for 12-16 hours, the oligomeric compounds, including withoutlimitation oligonucleotides and oligonucleosides, are recovered byprecipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligomeric compounds are analyzed by electrospray mass spectrometry(molecular weight determination) and by capillary gel electrophoresis.The relative amounts of phosphorothioate and phosphodiester linkagesobtained in the synthesis is determined by the ratio of correctmolecular weight relative to the −16 amu product (+/−32+/−48). For somestudies oligomeric compounds are purified by HPLC, as described byChiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtainedwith HPLC-purified material are generally similar to those obtained withnon-HPLC purified material.

Example 1 Preparation of Compound 10

Compound 1 was prepared according to published procedures by Steffens etal., Helvetica Chimica Acta, 1997, 80, 2426-2439 and was obtained as ananomeric mixture (α:β=4:1). Alkylation with ethyl iodoacetate yieldedcompound 2 as a mixture of 4 isomers, that was subsequently convertedinto the silylenol ether by treatment with LiHDMS and TBDMS-Cl at −78°C. At this stage the anomeric mixture was separated by columnchromatography. The α-anomer of 3 was treated with Et₂Zn to give 4 inabout 40% yield, together with about 30% of the corresponding epimericcyclopropane. Compound 4 was then converted into glycal 5 by treatmentwith TMS-triflate. NIS mediated nucleosidation of 5 with persilylatedthymine yielded stereospecifically the iodonucleoside 6 that wassubsequently deiondinated to the tricyclic nucleoside 7 by radicalreduction with Bu₃SnH. Removal of the silyl protecting groups with HF inpyridine afforded compound 8 that was subsequently tritylated andphosphitylated to give the desired phosphoramidite 10. All thestructures were confirmed by spectral analysis.

Example 2 Preparation of Compound 15

Compound 7 was prepared as illustrated in Example 1. Basic hydrolysis of7 with KOH afforded acid 11 that was converted into compound 12 bytreatment with Fmoc protected aminopropanol and the condensing agentEDC. Removal of the silyl protecting groups followed by tritylation andphosphitylation yielded the desired phosphoramidite 15. All thestructures were confirmed by spectral analysis.

Example 3 Preparation of Compound 19

Compound 11 was prepared as illustrated in Example 2 and was esterifiedto compound 16 with hexadecanol and EDC as condensing agent.Desilylation with HF followed by tritylation with DMT-Cl andphosphitylation lead to the desired phosphoramidite 19. All thestructures were confirmed by spectral analysis.

Example 4 Preparation of compound 21

Compound 11 was prepared as illustrated in Example 2 and was convertedto compound 20 with the singly protected 1,3-diaminopropane and EDC ascondensing agent. Desilylation with HF afforded compound 21. All thestructures were confirmed by spectral analysis.

Example 5 Preparation of Oligomeric Compounds

Following synthetic procedures well known in the art, some of which areillustrated herein, oligomeric compounds are prepared having at leastone tricyclic nucleosides, using one or more of the phosphoramiditecompounds illustrated in the Examples such as DMT phosphoramidites (seeCompound 10, Compound 15 or Compound 19).

Example 6 Preparation of Oligomeric Compounds for Tm Study

Following standard automated DNA synthesis protocols oligomericcompounds were prepared comprising one or more tricyclic nucleosides forTm studies. After cleavage from the solid support, the oligomericcompounds were purified by ion exchange HPLC and analyzed by LCMS usingstandard procedures. The Tm of the modified 10mer oligomeric compoundswere compared to an unmodified 10mer DNA oligonucleotide when duplexedto either DNA or RNA.

Tm's were determined using a Cary 100 Bio spectrophotometer with theCary Win UV thermal program was used to measure absorbance vs.temperature. For the T_(m) experiments, the oligomeric compounds wereprepared at a concentration of 1.2 M in a buffer of 150 mM NaCl, 10 mMphosphate, 0.1 mM EDTA at pH 7. The concentration determined at 85° C.was 1.2 M after mixing of equal volumes of selected oligomeric compoundand complementary RNA or DNA. The oligomeric compounds were hybridizedwith a complementary RNA or DNA by heating the duplex to 90° C. for 5minutes and then cooling to room temperature. T_(m) measurements weretaken using a spectrophotometer while the duplex solution was heated ina cuvette at a rate of 0.5° C./min starting at 15° C. until thetemperature was 85° C. T_(m) values were determined using Vant Hoffcalculations (A₂₆₀ vs temperature curve) using non self-complementarysequences where the minimum absorbance related to the duplex and themaximum absorbance related to the non-duplex single strand are manuallyintegrated into the program.

SEQ ΔTm/mod ΔTm/mod ID (° C.) (° C.) NO. Sequence (5′ to 3′) vs DNAvs RNA A01 AACTGTCACG  0  0 A02 AACTGT_(b)CACG −0.9 +0.4 A03AACTGT_(d)CACG +0.5 +2.1 A04 AACT_(b)GTCACG +0.1 +2.4 A05 AACT_(d)GTCACG+0.4 +2.4 A06 AACT_(b)GT_(b)CACG −0.7 +0.5 A07 AACT_(c)GT_(c)CACG −1.0+1.3 A08 AACT_(d)GT_(d)CACG −0.6 +1.2 A09 AACT_(e)GT_(e)CACG −13.0 −10.2A10 AACT_(f)GT_(f)CACG −2.0 +1.3 A11A_(a)A_(a)C_(a)T_(a)G_(a)T_(a)C_(a)A_(a)C_(a)G_(a) +1.3 +2.1 A12A_(a)A_(a)C_(a)T_(a)G_(a)T_(d)C_(a)A_(a)C_(a)G_(a) −2.8 −0.6 A13A_(a)A_(a)C_(a)T_(d)G_(a)T_(a)C_(a)A_(a)C_(a)G_(a) −3.8 −2.6 A14A_(a)A_(a)C_(a)T_(b)G_(a)T_(b)C_(a)A_(a)C_(a)G_(a) +1.1 +2.0 A15A_(a)A_(a)C_(a)T_(c)G_(a)T_(c)C_(a)A_(a)C_(a)G_(a) +1.1 +2.0 A16A_(a)A_(a)C_(a)T_(d)G_(a)T_(d)C_(a)A_(a)C_(a)G_(a) +1.1 +2.3

The Tms of the unmodified oligomeric compound A01 are 47.9° C. and 48.3°C. duplexed with DNA or RNA respectively. Each internucleoside linkinggroup is a phosphodiester. Each nucleoside not followed by a subscriptis a β-D-2′-deoxyribonucleoside and each nucleoside followed by asubscript “a” to subscript “f” are as defined below.

Example 7 Preparation of Oligomeric Compounds for Uptake Studies intoHeLa Cells

Hela cells were grown at 37° C. in Dulbecco's Modified Eagle's Medium(DMEM, Invitrogen) supplemented with 10% (v/v) Fetal Calf Serum(Amimed), 100 units/ml penicillin (Invitrogen) and 100 μg/mlstreptomycin (Invitrogen). For transfection experiments, 1×10⁵ cellswere seeded in duplicate in six-well plates, half of them containingcover slips, 24 h before transfection. Then, the medium was replaced bya solution of oligonucleotide (10 μM final concentration) having thesequence 5′-T-t-T-t-T-t-T-t-T-t-FAM-3′ where T is deoxythymidine and tis either tc^(ee)T (sunscript b of example 6), tc^(hd)T (subscript e ofexample 6) or tcT (subscript a of example 6) and FAM is6-carboxyfluorescein, in DMEM+/+ (FCS, P/S).

The transfection medium was removed after 48 h at 37° C. and cells werewashed with 2×1 ml PBS and resuspended in 1 ml fresh DMEM+/+. Fixationof the cells on the cover slips was carried out using a solution ofparaformaldehyde (1 ml, 3.7% in PBS) for 10 min followed by washing withPBS (2×1 ml), permeabilization of the cell membrane with Triton x-100(0.2%, Promega) for 10 min and washing with PBS (2×1 ml). The coverslips were treated with a few drops of polyvinylalcohol (Mowiol) andnuclear stain 40,60-diamidino-2-phenylindole (DAPI). Cells were analyzedby fluorescence microscopy (Leica DMI6000 B, Leica Microsystems;software: Leica Application Suite) 48 h post transfection.

The microscopy pictures show strong fluorescein fluorescence in thecytosol of cells treated with oligonucleotides containing tc^(hd)T(subscript e of example 6), while no fluorescence is observed whenoligonucleotides containing tc^(ee)T (sunscript b of example 6) or tcT(subscript a of example 6) were used

Example 8 Preparation of Oligomeric Compounds for Uptake Studies intoHEK293T Cells

HEK293T cells were grown at 37° C. in Dulbecco's Modified Eagle's Medium(DMEM, Invitrogen) supplemented with 10% (v/v) Fetal Calf Serum(Amimed), 100 units/ml penicillin (Invitrogen) and 100 μg/mlstreptomycin (Invitrogen). For transfection experiments, 2×10⁵ cellswere seeded in duplicate in six-well plates, half of them containingcover slips, 24 h before transfection. Then, the medium was replaced bya solution of oligonucleotide (10 μM final concentration) having thesequence 5′-T-t-T-t-T-t-T-t-T-t-FAM-3′ where T is deoxythymidine and tis either unmodified deoxythymidine, tc^(ee)T (sunscript b of example6), tc^(hd)T (subscript e of example 6) or tcT (subscript a of example6) and FAM is 6-carboxyfluorescein, in DMEM+/+(FCS, P/S). Thetransfection medium was removed after 48 h at 37° C. and cells werewashed with 2×1 ml PBS and resuspended in 1 ml fresh DMEM+/+. Fixationof the cells on the cover slips was carried out using a solution ofparaformaldehyde (1 ml, 3.7% in PBS) for 10 min followed by washing withPBS (2×1 ml), permeabilization of the cell membrane with Triton x-100(0.2%, Promega) for 10 min and washing with PBS (2×1 ml). The coverslips were treated with a few drops of polyvinylalcohol (Mowiol) andnuclear stain 40,60-diamidino-2-phenylindole (DAPI). Cells were analyzedby fluorescence microscopy (Leica DMI6000 B, Leica Microsystems;software: Leica Application Suite) 48 h post transfection.

The microscopy pictures show strong fluorescein fluorescence in thecytosol of cells treated with oligonucleotides containing tc^(hd)T(subscript e of example 5), while no fluorescence is observed whenoligonucleotides containing tc^(ee)T (subscript b of example 5) or tcT(subscript a of example 5) were used.

1. A tricyclic nucleoside described by general Formula I:

wherein: Bx is a heterocyclic base moiety; one of T¹ and T² is hydroxyl(—OH) or a protected hydroxyl and the other of T¹ and T² is a phosphateor a reactive phosphorus group; q¹ and q² are each, independently, H, For Cl, at least one of q³, q⁴ and q⁵ is, independently, a groupdescribed by a general formula -A¹-X_(h)-A²-Y_(n), wherein A¹ is aC_(k)-alkyl, C_(k)-alkenyl or C_(k)-alkynyl, with k being an integerselected from the range of 0 to 20, X_(h) is —C(═O)—, —C(═O)O—,—C(═O)NR—, —O—, —S—, —NR—, —C(═O)R, —C(═O)OR, —C(═O)NR₂—, —OR, —SR or—NR₂, with each R being selected independently from H, methyl, ethyl,propyl, butyl, acetyl and 2-hydroxyethyl, and h is 0 or 1, A² is aC_(i)-alkyl, C_(i)-alkenyl or C_(i)-alkynyl, with i being an integerselected from the range of 0 to 20, Y is a substituent group attached toany carbon atom on A¹ and/or A², selected from —F, —Cl, —Br, ═O, —OR,—SR, —NR₂, —NR₃ ⁺, NHC(═NH)NH₂, —CN, —CN, —NC, —NCO, —NCS, —SCN, —COR,—CO₂R, CONR₂.—R, with each R being selected independently from H,methyl, ethyl, propyl, butyl, acetyl and 2-hydroxyethyl, and n is 0, 1,2, 3, 4, 5 or 6, wherein k+i equals at least 1, the other ones of q³, q⁴and q⁵ are, independently, H, F or Cl, one of z¹ and z² is H and theother of z¹ and z² is H, —OH, F, Cl, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃,OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂.
 2. The tricyclicnucleoside of claim 1, wherein Bx is uracil, thymine, cytosine,5-methylcytosine, adenine or guanine.
 3. The tricyclic nucleoside ofclaim 1, wherein T¹ is hydroxyl or protected hydroxyl, and wherein T² isa reactive phosphorus group selected from an H-phosphonate or aphosphoramidite.
 4. The tricyclic nucleoside of claim 1, wherein T¹ is4,4′-dimethoxytrityl and T² is diisopropylcyanoethoxy phosphoramidite ora controlled pore glass surface.
 5. The tricyclic nucleoside of claim 1,wherein q³ is described by the general formula -A¹X_(h)-A²-Y_(n), and q⁴and q⁵, independently of each other, are H, F or Cl.
 6. The tricyclicnucleoside of claim 1, wherein k is an integer from 3 to 16, h is 0, iis 0 and n is 1, 2, or
 3. 7. The tricyclic nucleoside of claim 1,wherein k is 1, h is I and X is —O—, COO—, CONH— or CONR—, i is aninteger from 3 to 16 and n is 1, 2, or
 3. 8. The tricyclic nucleoside ofclaim 1, wherein n is 1 and Y is selected from NH₂, NHR, NR₂, NR₃ ⁺ andNHC(═NH)NH₂, with R having the meaning defined above.
 9. The tricyclicnucleoside of claim 1, wherein Y is in the ω-position.
 10. The tricyclicnucleoside of claim 1, wherein the sum of i+k is an integer from 3 to16, particularly from 3 to 12, more particularly from 5 to
 10. 11. Thetricyclic nucleoside of claim 1, wherein A¹ is CH₂, h is land X_(h) is—C(═O)O—, —C(—O)NH—, and A² is a C2 to C16 alkyl, Y_(n) is NH₂ and n is0 or
 1. 12. The tricyclic nucleoside of claim 1, wherein A¹ is CH₂. 13.The tricyclic nucleoside of claim 1, wherein k is
 0. 14. A tricyclicnucleoside selected from the group of a.

b.

c.

d.

e.

f.

wherein Bx is selected from uracil, thymine, cytosine, 5-methylcytosine,adenine and guanine.
 15. An oligomeric compound comprising at least onetricyclic nucleoside according to claim 1, wherein said oligomericcompound comprises from 8 to 40 monomeric subunits.
 16. The oligomericcompound of claim 15, wherein each Bx is, independently, uracil,thymine, cytosine, 5-methylcytosine, adenine or guanine.
 17. Theoligomeric compound of claim 15, wherein each internucleoside linkinggroup is, independently, a phosphodiester internucleoside linking groupor a phosphorothioate internucleoside linking group.
 18. The oligomericcompound of claim 15 to 17, comprising a first region having at leasttwo contiguous tricyclic nucleosides having Formula II.
 19. Theoligomeric compound of claim 18 comprising a second region having atleast two contiguous monomeric subunits wherein each monomeric subunitin the second region is a modified nucleoside different from thetricyclic nucleosides of Formula II of said first region.
 20. Theoligomeric compound of claim 19 further comprising a third regionlocated between said first and second regions wherein each monomersubunit in the third region is independently, a nucleoside or a modifiednucleoside that is different from each tricyclic nucleoside of FormulaII of the first region and each monomer subunit of the second region.21. The oligomeric compound of claim 15 comprising a gapped oligomericcompound having an internal region of from 6 to 14 contiguous monomersubunits flanked on each side by an external region of from 1 to 5contiguous monomer subunits wherein each monomer subunit in eachexternal region is a tricyclic nucleoside of Formula II and each monomersubunit in the internal region is, independently, a nucleoside or amodified nucleoside.
 22. The oligomeric compound of claim 15, comprisingone or several nucleotide blocks selected from a.

b.

c.

wherein T³ and T⁴ have the meanings outlined above.
 23. A method forsolid-phase synthesis of an oligonucleotide comprising the use of atrycyclic nucleoside according to claim 1.